Microwave-assisted biodiesel production using –SO3H functionalized heterogeneous catalyst derived from a lignin-rich biomass

The synthesis of biodiesel from renewable resources has immense potential as a sustainable and cost-effective energy alternative. In this work, a reusable –SO3H functionalized heterogeneous catalyst that has a total acid density of 2.06 mmol/g was prepared from walnut (Juglans regia) shell powder by low-temperature hydrothermal carbonization (WNS-SO3H). Walnut shell (WNS) contains more lignin (50.3%), which shows great resistance toward moisture. The prepared catalyst was employed for the effective conversion of oleic acid to methyl oleate by a microwave-assisted esterification reaction. The EDS analysis revealed the significant presence of sulfur (4.76 wt%), oxygen (51.24 wt%), and carbon (44 wt%) content. The results of the XPS analysis confirm the bonding of C–S, C–C, C=C, C–O, and C=O. Meanwhile, the presence of –SO3H (the responsible factor for the esterification of oleic acid) was confirmed by FTIR analysis. Under the optimized conditions (9 wt% catalyst loading, 1:16 oleic acid to methanol molar ratio, 60 min reaction time, and 85 °C temperature), the conversion of oleic acid to biodiesel was found to be 99.01 ± 0.3%. The obtained methyl oleate was characterized by employing 13C and 1H nuclear magnetic spectroscopy. The conversion yield and chemical composition of methyl oleate were confirmed by gas chromatography analysis. In conclusion, it can be a sustainable catalyst because the catalyst preparation controls the agro-waste, a great conversion is achieved due to the high lignin content, and the catalyst was reusable for five effective reaction cycles.

www.nature.com/scientificreports/ they were obtained without additional purification. To get distilled water, the Simplicity® UV Water Purification System (Merck) has been used.
Preparation of catalyst. Walnut shells were rinsed with distilled water and dried in a hot air oven for 30 h at a temperature of 80 °C. Oven-dried walnut shells are then grounded into powder using an electric grinder. The walnut shell powder was sieved through a 125-micron stainless steel sieve. After that, the measured amount of walnut powder was taken in four different borosilicate bottles (2 g in each bottle). Different proportions of concentrated sulphuric acid 1:10, 1:13, 1:17, and 1:20 (10.62, 13.82, 18.05, and 21.27 mL respectively) were added in the borosilicate bottles, and the resultant mixture was stirred for 30 min to make the homogeneous solution.
After that, these bottles were kept in the oven at 80 °C for 24 h. The final mixture is cooled down and diluted with distilled water and washed multiple times to remove excess sulfate. The presence of sulfate ions was checked using a BaCl 2 solution. After washing, the catalyst was dried in the oven at 80 °C for 12 h (Fig. 1). The synthesized catalysts were stored in airtight vials and named WNS-1 (1:10), WNS-2 (1:13), WNS-3 (1: 17), and . Further CHNS (Carbon, Hydrogen, Nitrogen, and Sulfur) elemental analyses were performed to determine the elemental composition of the prepared catalyst (Table 1). SO 3 H is the most important component for catalyst activity in the esterification reaction. Consequently, WNS-4 was taken to perform the esterification of oleic acid and renamed WNS-SO 3 H due to the high acid density. CHNS analysis. The CHNS analysis of WNS-1, WNS-2, WNS-3, and WNS-SO 3 H catalysts was performed to determine the elemental composition. To perform the analysis 10 mg powder sample of each catalyst was taken in a tin capsule and placed in an autosampler. The tin boat enclosing the sample falls into the reactor chamber where excess oxygen was introduced before (at about 1150 °C). The complete oxidation is reached with a tungsten trioxide catalyst which is passed by the gaseous reaction products (CO 2 , H 2 O, NO 2, NO 3 . and SO 2 ). The product gas mixture flows through a silica tube packed with copper granules. In this zone at 850 °C excess oxygen is bound and nitric/nitrous oxides are reduced. The leaving gas stream includes CO 2 , H 2 O, N 2 , and SO 2 , and was adsorbed at appropriate traps. High-purity helium was used as carrier gas. Finally, the gas mixture was passed to a gas chromatographic system. Separation of the elemental species (carbon, hydrogen, nitrogen, and sulfur) was done and detected using Thermal Conductivity Detector.
Determination of acid density. Acid-base back titration was performed to determine the total acid density of the synthesized catalyst 24 . The following steps are used to calculate the total acid density of the catalysts; In a 50 mL conical flask, 0.05 g of each catalyst was mixed with 2 M aqueous solution of NaCl (15 mL). The resultant mixture is sonicated for 30-min using an ultrasonic sonicator to sonicate the suspension. After that, the solution was filtered, and the filtrated solution was titrated with an aqueous solution of 0.02 M NaOH solution using a   41,42 . Each titration result has been triplicated and their concordant value is noted. The formula for calculating the acid concentration is as follows: Here, OH − is the NaOH concentration, m is the catalyst mass, and V is the titrant volume. The acid density of all four catalysts has been checked using the same procedure and it has been found that WNS-4 (1:20) has the highest acid density 2.06 mmol/g.

Characterization of WNS-SO 3 H. Powder X-ray diffraction (XRD) spectroscopy, Fourier Transform
Infra-Red (FTIR), Spectroscopy, scanning electron microscopy (SEM), X-ray energy dispersion spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and CHNS analyzer, were used to characterize the WNS-SO 3 H catalyst. The ELEMENTAR vario EL III device was employed as a CHNS analyzer to determine the percentage amount of carbon, hydrogen, nitrogen, and sulfur. The XRD pattern in the catalyst was studied in a Philips X'pert Pro diffractometer equipped with Cu-K with a scanning rate of 2° min −1 and a range of 2θ between 10° and 90°. The functional groups in the catalyst were identified using FT-IR spectroscopy. For FT-IR spectroscopy in the 4000-400 cm −1 range, a Vertex 80 equipped with a Bruker 3000 Hyperion Microscope spectrometer and KBr pellets was used. For SEM-EDS; Jeol 639OLA/OXFORD XMX N was employed to look over the surface morphology of the WNS-SO 3 H. SEM-EDS was performed using an acceleration voltage of 0.5-60 eV and an EDAX detector area of 30 mm 2 . The XPS was performed on a Thermofisher Scientific Nexsa base model equipped with Auger electron microscopy, which has an ionization energy range of 21.2-40.8 eV. The TGA analysis was performed using TGA-DTA Hitachi STA7000 in the temperature range of 30-600 °C.

Activity evaluation of WNS-SO 3 H.
To evaluate the catalytic activity of WNS-SO 3 H, microwave-assisted esterification was performed. In a 15 mL ace pressure tube, WNS-SO 3 H catalyst (5-11 wt%) was taken in a homogenous mixture of oleic acid:methanol molar ratio (1:12-1:24) using 50 W microwave power at temperature (65-95 °C) using reaction time (60-100 min) to produce methyl oleate as a product (biodiesel) (Scheme 1). The optimum reaction conditions were catalyst dose 9 wt% (0.025 g), oleic acid to methanol molar ratio 1:16 (oleic acid 1 mmol (0.282 g):methanol 16 mmol (0.512 g), 85 °C temperature, and 60 min reaction time. Because the esterification reaction is an endothermic process, increasing the reaction temperature to an optimal value enhances biodiesel production by improving the rate of the reaction and shortening the reaction time 12 . The esterification reaction was carried out using a microwave (CEM, DISCOVER SYSTEM, DY 1073, 180/264 VAC.). Using the initial substrates and precoated silica gel (60 F254-Merck) plates, TLC (thin-layer chromatography) was employed to check the reaction's progress. After the reaction, the catalyst was isolated from the reaction mixture using filtration by Whatman paper, and methanol was employed to rinse the catalyst to remove impurities. A rotary evaporator was used to remove excess methanol from the reaction mixture and extract pure biodiesel.

Characterization of biodiesel.
After the extraction of pure biodiesel from the rotary evaporator under optimal conditions, a considerable amount of biodiesel is characterized using 1 H and 13 C nuclear magnetic resonance spectroscopy to confirm the formation of biodiesel (methyl oleate). 1 H and 13 C nuclear magnetic resonance spectroscopy was carried out in a Jeol instrument having model number ECZ500R/S1 having a frequency ranging from 10 to 535 MHz. The conversion of oleic acid into methyl oleate is obtained using Eq. (2).
In this equation (Eq. 1), A OMe stands for the integration of the OMe groups in FAME, and A CH2 stands for the total integration of the -CH 2 (OA + FAME).
(1) www.nature.com/scientificreports/ In order to verify the findings of the NMR investigation, a GC (gas chromatography) analysis was performed. GC was carried out using an Agilent model 8890 equipped with split/splitless injectors, polar columns (HP-5), and a UI Agilent column having diameter 30 m × 0.320 mm. The temperature of the oven was raised from 300 to 400 °C where the injection temperature was 300 °C. An external calibration curve has been prepared by methyl oleate (purchased from Merck) to quantify the peaks.
Catalyst leaching and reusability. To look over the reusability of the WNS-SO 3 H catalyst, the esterification process was carried out by following the optimum conditions (9 wt% WNS-SO 3 H loadings, 1:16 oleic acid:methanol molar ratio, 85 °C reaction temperature, and a reaction period of 60 min) for the five effective cycles of reaction. After every cycle of esterification, the catalyst was separated from the mixture using filtration and rinsed using methanol for 4 times to eliminate the impurities from the surface of the catalyst. After that, the catalyst was oven dried at 80 °C for 4 h and used in further cycles.
Leaching test of WNS-SO 3 H catalyst performed by mixing catalyst with methanol solution. The solution is stirred using a magnetic stirrer for 6 h. After this, the solution was filtered and the resultant methanol solution is added with oleic acid under the optimum condition (85 °C reaction temperature, and a reaction period of 60 min).

Kinetics of reaction.
The chemical kinetics of WNS-SO 3 H catalyzed esterification of oleic acid with methanol can be explained using the following assumptions.
1. The rate of reaction of esterification of oleic acid was regulated by chemical reaction. 2. Since methanol is used in excess to carry out the reaction, its concentration may be taken to be constant. 3. The equilibrium of the reaction is shifted in the direction of FAME production because of the high concentration of methanol.
Therefore, the esterification is assumed to be a pseudo-first-order reaction 24,43 . The rate of the reaction between oleic acid and methanol can be shown by Eq. (3).
where k = rate constant, t = reaction time, and [oleic acid] represents the oleic acid concentration. To determine the first-order rate constant by monitoring methyl ester conversion t was varied in Eq. (4).
The activation energy of the esterification reaction was calculated at different temperatures: 55, 65, 75, and 85 °C. The Arrhenius Eq. (5) was used to calculate the energy of activation at different temperatures (55-85 °C) 44 .

Results and discussion
Powder X-ray diffraction analysis. Figure 2 depicts the XRD pattern of all synthesized catalysts including the WNS-SO 3 H catalyst. Broad diffraction peaks of the catalysts suggest the amorphous nature. The large diffraction peaks obtained at 2θ = 20°-28° are responsible for the amorphous carbon structures that comprise the random orientation of aromatic carbon sheets 3,11 . A weak broad diffraction peak at 2θ = 43° represents the presence of a small amount of graphite carbon 12 . The analysis reveals that sulfonation (different molar ratios of sulfuric acid used for catalyst synthesis) does not affect the structure of the carbon skeleton, consequently, the carbonaceous structure of all prepared catalysts is the same. X-ray photoelectron spectroscopy. XPS analysis was performed to look into the chemical valences of the surface group on the surface of WNS-SO 3 H. Figure 3a shows the carbon spectrum, which consists of three peaks at 284.5, 286.7, and 288.9 eV, which is related to C=C, C-S, and C=O bonds respectively 25 . The oxygen spectrum shown in Fig. 3b was found to have two peaks at 531.9 eV related to C-O and 533.9 eV related to C=O. Figure 3c represents the sulfur spectra with two peaks at 167.3 and 168.8 eV, which represent the presence of C-SO 2 -C and C-SO 3 H bonding 25 . Furthermore, Fig. 3d displays the overall XPS spectrum of the catalyst WNS-SO 3 H. Therefore, the XPS analysis indicates that carbon, oxygen, and sulfur were present in the catalyst and also confirms that most of the sulfur is present as -SO 3 H, however, some content is also found in the form of SO 2 .
SEM-EDS analysis. Scanning electron microscopy and X-ray energy dispersion spectroscopy were performed on WNS-SO 3 H to evaluate the structural morphology and composition of elements, respectively. SEM images in Fig. 4a-c are corresponding to the structural morphology of WNS-SO 3 H at different magnifications. SEM images reveal the non-pores and irregular morphology of the catalyst. The insertion of -SO 3 H groups block the surface pores resulting in the non-pores structure and showing the effective sulfonation of the catalytic surface. The surface morphology of the WNS-SO 3 H catalyst is similar to the previously reported studies that favor the effectiveness of the catalyst 45,46 . Additionally, EDS analysis was carried out to determine the elemental composition of the catalyst. Figure 4d reveals the composition of elements (Carbon 44 wt%, Oxygen 51.24 wt%,   www.nature.com/scientificreports/ represents the C=C bonding of polyaromatic skeletal. The peak at 1690 cm −1 is related to the C=O bond from the -COOH group 25 . These findings corroborate the existence of acidic groups -COOH and -SO 3 H groups. The -SO 3 H groups substitute hydrogen on the solid surface to covalently attach to the carbon structure 47 . The peak at 3368 cm −1 is related to the -OH group existing at the catalyst's surface. Thermogravimetric analysis. The TGA analysis of the WNS-SO 3 H catalyst is shown in Fig. 6. The WNS-SO 3 H catalyst gives gradual weight loss with two decomposition stages at 150 and 250-300 °C. A significant weight loss was observed at 150 °C, which might be due to the removal of water content. The second decomposition stage of the catalyst occurred between 250 and 300 °C, which reveals the loss of -SO 3 H groups and cellulose degradation in the form of moisture (H 2 O) and gases (NH 3 and CO 2 ). After 300 °C weight loss in the WNS-SO 3 H catalyst was gradual, which was due to the lignin decomposition 48 .  To evaluate the effect of catalyst dosage, the catalyst concentration was regulated from 5 to 11 wt% in relation to the mass of oleic acid. It has been observed that when the catalyst concentration was raised from 5 to 9 wt%, there was a continuous increment in conversion, demonstrating that the quantity of catalyst had a substantial impact on the conversion of oleic acid. Because as the catalyst concentration increases, sulfur content also increases, which provides more reaction sites that result in high conversion. Nevertheless, when catalyst loading was increased by more than 9 wt% showed slightly reduced conversion (Fig. 7a). Since esterification is a reversible reaction, therefore an excess amount of catalyst may promote the reversible esterification reaction, consequently, overall product efficiency has been decreased 31,49 . Therefore, 9 wt% catalyst concentration has been taken as the optimum dose of the catalyst.
Oleic acid:methanol ratio. Esterification is a reversible reaction, and to initiate the reaction, an excess amount of methanol is frequently used to increase the solubility of the fatty phase with the phase of methanol. Furthermore, an excess of methanol may not result in improved catalytic efficiency since a considerable quantity of alcohol might limit oleic acid accessibility to the active sites of the catalyst. Therefore, a desired molar ratio of methanol is required for the esterification to carry out the reaction in a forward direction 27,30 . The impact of oleic acid to methanol molar ratio on conversion yield was investigated by changing the molar ratio to 1:12, 1:16, 1:20, and 1:24, at 85 °C for 60 min and 9 wt% catalyst doses. As the ratio of oleic acid to methanol went from 1:12 to 1:16, the conversion yield increases intensely. However, when the ratio went from 1:16 to 1:20, there was no significant change. which may be ascribed to reaction phase equilibrium. Furthermore, on increasing the ratio to 1:24, it has been observed that the conversion yield was decreased (Fig. 7b). Therefore, a 1:16 molar ratio was determined as the optimum oleic acid:methanol for the esterification reaction, and further reactions were performed with this ratio. It is worth mentioning that lower ratios, such as 1:16, may be used to achieve significant conversion without adversely affecting the activity of the catalyst. Because less alcohol is needed to drive the reaction and manufacturing costs would be also reduced 50 .
Reaction temperature. To look over the temperature effect on the esterification reaction, the temperature was changed from 65 to 95 °C. In the meantime, the remaining parameters were fixed at 9 wt% catalyst loading, a 1:16 oleic acid:methanol molar ratio, and 60 min of reaction time. A remarkable increment in conversion yield was noticed when the temperature was raised from 65 to 75 °C and reached equilibrium at 85 °C. By enabling mass transfer between the reactants and the surface of the catalyst, the molecules' increased kinetic energy causes the reaction to proceed more quickly as the temperature rises. The conversion was reduced by very high temperatures because they accelerate the methanol evaporation, which reduces the quantity of methanol that is present for the reaction with oleic acid 51 . As a result, conversion yield diminished with a further increase in temperature to 95 °C (Fig. 7c). Consequently, it was determined that the optimum temperature for the reaction was 85 °C. The reaction temperature for the esterification process is very less in comparison to similar studies, that used 100-150 °C reaction temperature 52,53 .
Reaction time. Time is a determining factor that greatly influences the catalyst's activity during biodiesel formation 52 . Reaction times were changed from 60 to 100 min to explore the effect of time on esterification reactions. However, other parameters were fixed at 9 wt% catalyst loading, a 1:16 oleic acid:methanol molar ratio, and 85 °C temperature. The highest yield of FAMEs, 99.01 ± 0.3% was found at a reaction time of 60 min (Fig. 7d).
There was hardly any refinement in biodiesel yield after increasing the reaction time from 60 to 100 min. This phenomenon is described by the reversible esterification reaction and can be initiated by allowing the reaction www.nature.com/scientificreports/ duration to exceed its optimum value. As a consequence, the formed biodiesel was subsequently hydrolyzed 54 . Prolonged reaction times have been shown to reduce surface area through a decrease in active sites 54 . Therefore, the optimum reaction condition was fixed at 9 wt% catalyst loading, a 1:16 oleic acid:methanol molar ratio, 85 °C temperature, and 60 min reaction time for the microwave-assisted biodiesel production using WNS-SO 3 H heterogeneous acid catalyst.

Comparative study of conventional and microwave-assisted biodiesel synthesis. The catalytic
performance of the WNS-SO 3 H catalyst was assessed by both conventional and microwave-assisted methods. The conventional method was used to perform the esterification reaction using the optimum conditions (9 wt% catalyst loading, a 1:16 oleic acid:methanol molar ratio, 85 °C temperature, and 60 min reaction time). For the conventional biodiesel synthesis, 9 wt% WNS-SO 3 H was taken in an ace pressure tube in 15 mL with a 1:16 molar ratio of oleic acid to methanol. The pressure tube was placed in a glycerol bath on a hot plate magnetic stirrer with the help of a holding stand. The conversion efficiency (%) obtained by conventional and microwave-assisted methods was 86.78 ± 0.6% and 99.01 ± 0.3%, respectively (Fig. 8). These findings are similar to the reported literature 26 . Furthermore, a comparison of the energy requirements of both the mechanically stirred reactor and the microwave-irradiated reactor was established. In our investigation, the power used by the microwave system during a 60 min reaction was 50 W, whereas, in a conventional mechanical stirred system, the same esterification reaction consumes 140 W of electricity over 60 min. As a result, the total energy used for the microwave-assisted esterification reaction was 90 W lower than the energy used for the mechanically stirred system, which meant that the consumption of energy in the microwave system would be even less. Furthermore, in comparison to previous studies, the energy and time consumption for the esterification of oleic acid with methanol in the presence of a WNS-SO 3 H catalyst is lower, resulting in higher conversion efficiency 55,56 .

Comparison with previously reported -SO 3 H functionalized heterogeneous catalysts.
Heterogenous acid catalysts show excellent catalytic activity for biodiesel production by the esterification process. Moreover, SFHCs are gaining much interest due to their excellent performance. Since most of the reported SFHCs were performed under conventional mechanically stirred esterification reactions. However, in this study, a microwave-assisted esterification process was carried out to achieve the highest yield of biodiesel (99.01 ± 0.3%). www.nature.com/scientificreports/ Table 2 represents the comparative study of the as-prepared WNS-SO 3 H catalyst, including specific parameters such as oleic acid:methanol molar ratio, reaction time, and reaction method. These significant parameters affect biodiesel production in terms of cost values. WNS-SO 3 H catalyst gives the highest biodiesel yield using a very low oleic acid:methanol molar ratio (1:16) and a very short reaction time compared to previously reported works 24,43 . The reaction time is very low compared to other studies employing microwave-assisted biodiesel production methods 55 .
Biodiesel characterization. Microwave-assisted esterification reaction of oleic acid using methanol was performed in the presence of a WNS-SO 3 H catalyst to produce biodiesel. The final product of the reaction (biodiesel) was obtained employing optimum reaction parameters (9 wt% catalyst loading, a 1:16 oleic acid:methanol molar ratio, 85 °C temperature, and 60 min reaction time). Biodiesel was characterized after employing the purification process using a rotary evaporator. NMR and GC analysis was carried out for the biodiesel characterization, as described in "Characterization of biodiesel"" section. 1 H and 13 C nuclear magnetic resonance spectroscopy was carried out in a Jeol instrument having model number ECZ500R/S1 having a frequency ranging from 10 to 535 MHz. The biodiesel sample was dissolved in CDCl 3 to perform the NMR analysis. Figure 9a and b portrayed the 1 H NMR and 13 C spectrum of biodiesel obtained by WNS-SO 3 H catalyzed esterification of oleic acid. The signal at 0.87 ppm is for the aliphatic -CH 3 group present at the end of the chain. The signals at 1.27 and 1.6 ppm are related to aliphatic -CH 2 groups away from double bonds and ester groups, while the signal at 2 ppm is for -CH 2 groups adjacent to double bonds and 2.27 ppm represents the -CH 2 groups (deshielded protons) adjacent to the carbonyl group of the ester. An intense peak at 3.6 ppm (Fig. 9a) reveals the presence of -OCH 3 protons which confirms the product formation (biodiesel). Further, the signal at 5.31 ppm reveals the presence of an unsaturated system (-CH=CH-CH=CH-) 40,62 . Figure 9b represents the 13 C NMR spectrum of synthesized biodiesel in which a signal at 14.1 ppm corresponds to the -CH 3 region. The signal in between 22.6 and 34.1 ppm revealing the -(CH 2 )n-region of biodiesel molecules. An intense signal at 51.5 ppm confirms the presence of the -COO-CH 3 region. The signal Figure 8. Comparative study of biodiesel synthesis, following conventional and microwave-assisted methods.  www.nature.com/scientificreports/ Figure 10 displayed the data obtained by GC analysis that confirms the biodiesel yield. The range of oven temperature during the GC analysis was 300-400 °C and pure methyl oleate was used as a standard.
Chemical kinetics of esterification reaction. The esterification of oleic acid is carried out by a WNS-SO 3 H catalyst in a homogeneous system where the overall reaction rate is controlled by the chemical reaction. By calculating the slope of − ln(1 − x) versus response time, we have calculated the perceptible rate constant (k). The resulting straight line is shown in Fig. 11a, and its R 2 value of 0.96-0.98 confirms the feasibility of the pseudo-first-order reaction 34 . The activation energy was calculated by fitting the rate constant in the Arrhenius equation i.e., the value of lnk versus 1/T gives the activation energy and its slope value gives the pre-exponential factor (E a /R). The results from Fig. 11b show that the activation energy is 54.428 kJ mol −1 and the value of the pre-exponential factor is 5.4 × 10 -6 min −1 . The activation energies varied between 24.7 and 84.1 kJ mol −1 are appropriate for esterification reactions as mentioned in the literature 63,64 . Reusability of WNS-SO 3 H. Typically, the reusability of the WNS-SO 3 H catalyst has a significant impact on manufacturing costs independent of its catalytic capabilities. To assess the reusability of WNS-SO 3 H, the same catalyst was utilized five times in optimum reaction conditions. After every cycle, the catalyst was retrieved and rinsed with methanol 2-3 times and oven dried at 80 °C for 5 h before being reused again. A 99.01 ± 0.3% conversion was achieved in the first effective cycle of the esterification reaction using optimum conditions (9 wt% catalyst loading, a 1:16 oleic acid:methanol molar ratio, 85 °C temperature, and 60 min reaction time). For the next consecutive reaction cycles, the conversion obtained was 97.98%, 96.38%, and 88.24% for the 2nd, 3rd, and 4th cycles, respectively. Further, for the 5th cycle, conversion dropped significantly, and a 65.11% conversion yield was obtained. Figure 12 represents the results of catalyst reusability testing. After five reaction cycles, the catalyst became inactive as a result of the deactivation of the active sites (-SO 3 H groups) on the WNS-SO 3 H being  www.nature.com/scientificreports/ deactivated and resulting in decreased total acid density 26 . In this work, the author hypothesizes that the primary deactivation process was the leaching of -SO 3 H groups; several other studies also showed similar behavior. However, the efficiency of the catalyst in the fifth cycle is equivalent to that reported in the literature 25,65 . Araujo et al. 25 reported a significant decrease in the catalytic efficiency after 3 consecutive reaction cycles. After five reaction cycles the reused WNS-SO 3 H (RWNS) catalyst was characterized with XRD, FTIR, and SEM-EDS analysis to check out the structural, functional, and morphological changes. Figure 13a depicts the XRD pattern of RWNS and confirms that no structural changes occurred after the five consecutive reaction runs 11 . The FTIR spectra of RWNS (Fig. 13b) reveal the presence of all the functional groups -SO 3 H, O=S=O, C=O, C=C, and OH. However, the peaks related to these functional groups were slightly shifted from the peaks of fresh catalysts 24 . A decrease in the sulfur content from 4.76 to 2.27 wt% was observed by EDS analysis. Furthermore, the morphology of the catalyst was also changed and a pores structure can be seen in Fig. 13c. The removal of -SO 3 H sites promote the pores structure of the catalyst and favor the catalyst deactivation. Meanwhile, the total acid density of the WNS-SO 3 H catalyst decreased from 2.06 to 1.32 mmol/g after the fifth reaction cycle.

Conclusion
The WNS-SO 3 H catalyst was prepared by low-temperature hydrothermal carbonization using lignin-rich biomass (walnut shell powder). The as-prepared catalyst provides the highest conversion yield (99.01 ± 0.3%) of biodiesel through microwave-assisted esterification reaction under the optimal reaction conditions. The obtained results show that the utilization of waste walnut shells solves environmental problems and effectively deals with sustainable energy. The WNS-SO 3 H catalyst can be employed for sustainable, eco-friendly, and cheap-cost production of biodiesel followed by the esterification of oleic acid. Moreover, the high amount of walnut shell waste and better reusability of the catalyst makes the catalyst appropriate for large-scale production. www.nature.com/scientificreports/

Data availability
All data generated or analyzed during this study are included in this published article. www.nature.com/scientificreports/